Transcription initiation is a major point of regulation of cellular processes. The long-term goal of this application is to obtain a detailed understanding of all steps involved in initiation of transcription, a process in which DNA-dependent enzyme RNA polymerase first locates promoter and in subsequent isomerization step melts a segment of DNA duplex in the vicinity of transcription start point to expose the template strand of DNA. Understanding transcription at a molecular level will be important for advancing the basic knowledge of this fundamental cellular process. Additionally, bacterial transcription machinery is an attractive target for drug discovery due to a remarkable conservation of structural and functional properties among bacterial RNA polymerases. Understanding bacterial transcription will thus aid in discovery of new antibiotics, an important health related issue due to the increasing problems with drug resistant microorganisms. The proposal is focused on E. coli RNA polymerase a subunit that in recent studies was shown to play intimate roles in all steps of transcription initiation. Three aims addressing fundamental issues concerning the role of this subunit in finding and melting of promoter DNA by RNA polymerase will be pursued: Aim #1. To determine molecular mechanism of initiation of transcription bubble formation. Aim #2. To determine functional roles of sigma region 4 - beta subunit "flap" domain contact. Aim #3. To determine the relative importance of sigma - DNA contacts for promoter search by RNA polymerase. Molecular events of transcription initiation are complex and involve large multi component complexes. Thus, a multi disciplinary approach combining biophysical (fluorescence and mass spectroscopy), biochemical, and molecular biology methods will be used. It is expected that a detailed understanding of the role of several protein-protein and protein-DNA contacts in RNA polymerase function will be obtained.